The combined tumour-based Fascin/Snail and stromal periostin reveals the effective prognosis prediction in colorectal cancer patients

Colorectal cancer (CRC) is the third most common malignancy cause of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) promotes cancer metastasis and a tumour-based Glasgow EMT score was associated with adverse clinical features and poor prognosis. In this study, the impact of using the established five tumour-based EMT markers consisting of E-cadherin (E-cad), β-catenin (β-cat), Snail, Zeb-1, and Fascin in combination with the stromal periostin (PN) on the prediction of CRC patients’ prognosis were invesigated. Formalin-fixed paraffin-embedded tissues of 202 CRC patients were studies the expressions of E-cad, β-cat, Snail, Zeb-1, Fascin, and PN by immunohistochemistry. Individually, cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Ss), and stromal PN (Ps) were significantly associated with reduced survival. A combination of Ps with Fc, Fs, and Sn was observed in 2 patterns including combined Fc, Fs, and Ps (FcFsPs) and Fc, Sn, and Ps (FcSnPs). These combinations enhanced the prognostic power compared to individual EMT markers and were independent prognostic markers. As the previously established scoring method required five markers and stringent criteria, its clinical use might be limited. Therefore, using these novel combined prognostic markers, either FcFsPs or FcSnPs, may be useful in predicting CRC patient outcomes.


Introduction
Globally, colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the fifth cause of cancer-related mortality, followed by lung cancer, liver cancer, gastric cancer, and breast cancer [1].Although the prognosis of early-stage CRC patients has improved greatly due to the advanced detection and treatment, advanced CRC patients still exhibit a poor prognosis with a 5-year survival rate of less than 20%, due to the presence or development of metastasis [2].Approximately, 20% of CRC patients have metastasis at diagnosis and more than half of patients will develop metastases and relapse after initial treatment [2,3].
The epithelial-mesenchymal transition (EMT) is a process of phenotypic transformation where epithelial cells lose cell-cell junction and polarity to become more stem-like or mesenchymal cells [4].In cancer, the EMT process is believed to play a crucial role in treatment resistance and metastasis [4].There are several proteins involved in EMT such as E-cadherin (E-cad), β-catenin (β-cat), Fascin, Snail, Zeb-1, and periostin (PN) [5,6].Each of these markers has previously been reported to have prognostic value in CRC [6].In normal cells, membranous E-cad forms a complex with β-cat, preventing its nuclear translocation and the initiation of EMT [7].In the metastatic setting, downregulation of E-cad allows β-cat to translocate into the nucleus and promotes upregulation of EMT-related gene expressions such as Snail, and Fascin [8,9].PN is a matricellular protein found in the periosteum and involved in bone extracellular matrix remodeling [10].Aberrant expression of PN has been observed in various cancers and its expression correlates with shortened patient survival time [10].Studies have reported that PN derived from cancer-associated fibroblasts can promote cancer cell metastasis [11,12].
The prognostic value of individual EMT markers has long been recognized in CRC patients [9,[13][14][15][16].Previously, we have reported the tumour-based EMT score consisting of a combination of E-cad, β-cat, Snail, Zeb-1, and Fascin expression and the association of this score with the adverse clinical features and poor prognosis in Glasgow CRC patients [6].Furthermore, the expression of PN in the stroma of CRC tissues was correlated with the presence of metastasis and reduced survival time in CRC patients [17][18][19][20].The high expression of PN in stromal cells in CRC was reported to mediate both tumorigenesis and tumor progression via several signaling pathways [17,21,22].
Though these markers can independently stratify the prognosis of CRC patients, it is still unclear whether PN in combination with the previously reported five tumour-based EMT markers can enhance the prognostic power in CRC patients or enable EMT markers to be reduced in number for clinical utilization.In the current study, we investigated the impact of using the established five tumour-based EMT markers, stromal PN, and the combination of stromal PN, and EMT markers for better CRC patients' prognosis.Stromal PN in combination with cytoplasmic, and stromal Fascin, and nuclear Snail was associated with patients' survival.These findings could be translated to an immunohistochemistry staining panel including only Fascin and Snail in the tumour cells and stromal PN to aid in clinician decision-making.

Patient samples
Two hundred and two CRC specimens were identified retrospectively from Siriraj Hospital diagnosed which were the surgical samples from patients who signed the consent for surgical procedures between 2009 to 2015.Matched clinical data and formalin-fixed paraffin-embedded (FFPE) tissues were obtained under the collection protocol approved by Siriraj Hospital Institutional Review Board, COA no.Si 544/2015 and Si 628/2021.These surgical samples were then kept at Department of Pathology, Faculty of Medicine Siriaj Hospital as the hospital regulation.To use these samples, we asked for the permission from the hospital director through the content included in the process of ethical approval by the institutional review board.Patients who received neoadjuvant chemotherapy and/or died within 30 days of surgery were excluded from the study.

Immunohistochemistry scoring
The stained sections were scanned using Hamamatsu NanoZoomer (Welwyn Garden City, Hertfordshire, UK) at 20x magnification and visualized on NDP viewer (NanoZoomer Digital Pathology software, Hamamatsu Photonics K.K.).Staining for the five EMT markers was assessed by a single examiner (NJ), blinded to clinicopathological data, using a weighted histoscore [23], calculated as follows: (% tumour with no staining x 0) + (% tumour with weak staining x1) + (% tumour with moderate staining x 2) + (% tumour with strong staining x3), giving a range of scores between 0 and 300 for each marker at the membrane, cytoplasm, nucleus, and stroma compartments.Ten percent of sections were also co-scored by a coinvestigator (JE) and the interclass correlation coefficient was calculated to be > 0.7 for all markers.For PN staining, the scoring values were evaluated by the percentage of positive stromal cells (P) and the intensity of the staining signal (I).For P, 0-25%, 26-50%, 51-75%, and 76-100% were classified as grades 0, 1, 2, and 3.For I, unstained, slightly stained, intermediately stained, and strongly stained were classified as 0, 1, 2, and 3.The expression scoring was calculated by P x I which covered the total score of 0-9.Two investigators performed this scoring double-blind to the clinicopathological data and each other (CT and PT).

Statistical analysis
The expression of protein at each cellular compartment was divided into low and high using a publicly available R package in R-Studio to calculate the cut-off value for each protein.The correlation between protein expression and clinicopathological factors was accessed by Fisher's exact or Pearson's χ 2 test.The Kaplan-Meier log-rank test was used for the survival function.The univariate Cox regression was used to calculate the hazard ratio (HR) and 95% confidence interval (95% CI).The step-wise backward conditional Cox regression was used to identify the independent prognostic marker.All statistical analyses were performed in SPSS version 23.The P-value < 0.05 was considered statistically significant.

Results
Immunohistochemical staining of E-cad, β-cat, Fascin, Snail, Zeb-1 and stromal PN in CRC tissues E-cad, B-cat and Fascin expression was mainly observed in the membrane but some expression was also seen in the cytoplasm, nucleus and stroma (Fig 1A -1C).For Snail and Zeb-1, Expression was commonly observed in the cytoplasm, although nuclear and stromal expression was also detected (Fig 1D and 1E).The expression of PN was detected only in the stroma compartment (Fig 1F).

Demographic data and clinicopathological factor correlation
A total of 202 FFPE full sections from this Thai CRC patient cohort were included in this study.The number of sections stained for each marker was 148 for E-cad, Snail, and Zeb-1, 152 for β-cat, 192 for Fascin, and 202 for PN.
The demographic data for these sections were summarized (Table 1).The patient's ages ranged from 32 to 94 years with a median age of 64 years (Table 1).Around 54% of patients were younger than 65 years at the time of diagnosis with 56.4% of patients being male.The percentage of patients at stages I, II, III, and IV were 16.8%, 25.9%, 30.8%, and 26.5%, respectively.Local metastasis was present in 25.3% and distant metastasis was observed in 38.4% and 34.9% though lymphovascular and perineural routes.At the last follow up, 128 patients were alive, 57 patients were died by cancer-related death, and 17 patients were died by non-cancer related death.The median overall survival time was 49.9 months (range 1.5-104 months).

The combination of EMT markers and PN improves prognostic strength in CRC patients
The  For the FcSnPs model, patients were stratified into; 1) "good prognosis" which is either a low level of all markers (low Fc/low Sn/low Ps) or a high level of only one marker (high Fc/low Sn/ low Ps or low Fc/high Sn/low Ps or low Fc/low Sn/high Ps); 2) "intermediate prognosis" characterized by high expression of two markers (high Fc/high Sn/low Ps or high Fc/low Sn/high Ps or low Fc/high Sn/high Ps) (HR 6.944, 95% CI 3.629-13.287,p < 0.001),); and 3) "poor prognosis" characterized by high expression of all markers (high Fc/high Sn/high Ps) (HR 21.269, 95% CI 7.492-60.381,p < 0.001) (Fig 3B).Interestingly, both FcFsPs (HR 1.753, 95% CI 1.256-2.448,p = 0.001) and FcSnPs (HR 2.178 95% CI 1.097-4.322,p = 0.026) models demonstrated independent prognostic value for CRC patients (Table 3).

Discussion
The EMT process is considered one of the underlying mechanisms of CRC metastasis [24].During EMT, not only do the cancer cells undergo genotypic and phenotypic alteration but also the tumor microenvironment (TME) surrounding the cancer cell changes.The interplay between cancer cells and TME during EMT allows the cancer cells to metastasize [25,26].Metastatic CRC has a poor prognosis and is inherently more resistant to chemotherapy treatment [2,27].Therefore, predicting the metastatic event in CRC patients based on the changes in EMT and TME could help identify CRC patients who may require more aggressive treatment.In the present study, high expression of Fc, Sc, Sn, Ss, and Ps was individually associated with a reduced survival time in CRC patients.Furthermore, the combination of Fc, Fs, and Fs (FcFsPs) and Fc, Sn, and Ps (FcSnPs) could efficiently stratify prognosis for CRC patients.The combined markers demonstrated a higher prognostic strength when compared to using an individual marker and may provide a novel prognosis tool for CRC patients.
The expression of transcriptional factor Snail in cancer cells has long been recognized as a marker of poor prognosis for multiple malignancies including CRC [8,16,28].Snail is known as a negative regulator of E-cad at the transcriptomic level, an initiation event for cancer metastasis [29,30].Studies have reported that the presence of Snail in ovarian cancer contributes to immunosuppression by up-regulating CXCL1 and CXCL2 expression which promotes recruitment of myeloid-derived suppressor cells (MDSCs) [31,32].Furthermore, expression of Snail in esophageal squamous cell carcinoma also promotes PD-L1 expression that induces T cell apoptosis [31,32].The pro-tumorigenic function and prognostic value of Snail expression was also found in stromal cells [33][34][35].In CRC, Snail-expressing cancer-associated fibroblasts (CAFs) showed different cytokines secretion profiles including CCL1, CCL7, and CXCL1 when compared to normal fibroblasts affecting CRC cell migration [36].Furthermore, co-injection of Snail-knockout fibroblast and CRC cells demonstrated low proliferative index by ki67 staining, high expression of membrane E-cad in the CRC cells, and reduced local metastatic lesion when compared to co-injection with Snail-wild type fibroblast [36].Therefore, Snail expression has been identified in both cancer and stromal cells and both sources could function in concert to promote tumor progression.This evidence supports the findings in this study that high Sn predicts poor prognosis in CRC patients.
Fascin is an actin filament-bundling protein that is crucial for the maintenance of cell structure and secretory function of cells [37].In cancer, re-arrangement of the cytoskeleton is a prerequisite event before metastasis [9,37].Studies in CRC tissues demonstrated that Fascin expression was low in normal colon mucosa, but overexpressed in the cytoplasm of CRC cells [38,39].Moreover, the overexpression of Fascin in CRC is associated with distant metastasis, reduced survival time, and disease recurrence [39][40][41].These data support our findings that high Fascin in the cytoplasm of cancer cells is associated with poor prognosis of CRC patients.
In contrast to the expression in cancer cells, Fascin in the stroma of CRC was reported as a marker of good prognosis and that elevated expression inversely correlated with the stage of cancer and lymph node metastasis [42].This finding was consistent with the good prognostic value of stromal Fascin (Fs) observed in the present study.In endometrial neoplasia development, the expression of stromal Fascin was reduced while its increased expression was observed in endometrioid carcinoma; and the loss of stromal Fascin was also associated with a higher grade of endometrioid carcinoma [42].However, in the ovarian cancer model, knockdown or pharmacologic inhibition of Fascin in stromal cells was shown to reduce cell migration and may contribute to delayed metastatic events [43].Furthermore, a recent study using small molecule inhibitors specific to Fascin has shown that tumor-bearing mice treated with Fascin inhibitors and anti-program death-1 (PD-1) reduced tumor growth [44].Fascin inhibitors impede dendritic cell migration out from the tumor bed and enhance their antigen-uptake ability subsequently increasing T cells activation that controls tumor growth [44].Within tumor stroma, there are several types of cells and differential expression of Fascin in these cells may contribute to the different findings regarding the prognostic value of stromal Fascin and warrant further investigation.PN or osteoblast-specific factor 2 (OSF-2) is a secreted protein mainly found in osteoblast and functions in cell-matrix interactions, and cell differentiation [10].In CRC, PN was reported to be overexpressed in stromal cells and associated with aggressive clinical features and poor prognosis [18][19][20].Mechanistically, PN was found to activate integrin α5β1 or α6β4 and subsequently stimulate Akt/PI3K resulting in enhanced chemotherapy resistance, cell migration, and metastatic properties [17,21,45].Studies have demonstrated that PN secreted by stromal cells of various cancer can also modulate the expression of EMT-associated proteins such as β-cat, E-cad, and Zeb-1, supporting cancer metastasis [46][47][48].All of these data strongly support the finding that high PN in the stromal cells of CRC tissues is indicative of poor prognosis.Taken all together, the current study shows that by the following combinations of protein markers is predictive of poor prognosis in CRC patients, 1) low stromal Fascin/high both cytoplasmic Fascin and stromal PN (low Fs/high FcPs) and 2) high cytoplasmic Fascin/nucelar Snail/stromal PN (high FcSnPs).However, the potential approach could involve incorporating the FcFsPs/FcSn/Ps into the existing molecular classification framework.By assessing the expression levels of these markers alongside genetic signatures, a more comprehensive understanding of CRC subtypes and their prognostic implications may be achieved.Furthermore, considering the evident interaction between stromal and cancer cells highlighted in our study, there's a strong rationale for expanding the current classification system to encompass aspects of the tumor microenvironment.This could involve developing new classification schemes that incorporate both genetic and pathological signatures, thus providing a more holistic view of CRC biology and potentially refining treatment strategies.
Although individual EMT markers have prognostic value, the combination of multiple EMT markers has been reported as a superior tool for CRC clinical outcome stratification [6,49].In a previous study, Roseweir et al. demonstrated that by combing membrane Ecad, nuclear β-cat, and cytoplasmic Fascin, Snail, and Zeb-1 CRC patients could be stratified into three categories (absence, low, and high) of prognosis [6].However, based on this previous EMT scoring criteria, the number of samples categorized as absent or high was too low to perform statistical analysis.Additionally, as the scoring method required five markers and stringent criteria, its clinical use might be limited.The retrospective paraffinembedded tissues used in this study mostly had degraded mRNA from a long storage.This limitation restricts our ability to directly measure mRNA level.Moreover, the quantification of FcFsPs/FcSnPs to be guide the clinical practice is our future study.In comparison, the present study proposes a novel prognostic marker combination that requires only two or three markers to stratify CRC patients which could be more practical in the clinic.Both FcFsPs and FcSnPs models are proposed as independent prognosis factors and demonstrate superior prognostic strength.

Table 2 . Prognostic value of tumour-based E-cad, β-cat, Fascin, Snail, Zeb-1 and stromal PN in CRC samples.
https://doi.org/10.1371/journal.pone.0304666.t002 Ps with Fc, Fs, and Sn as FcFsPs and FcSnPs could efficiently stratify the prognosis of patients into good and poor prognostic groups (Fig 3Aand 3B).In the FcFsPs model, patients could be grouped into; 1) "good prognosis" characterized by either low expression of all markers (low Fs/low Fc/low Ps) or high Fs with either low expression of both Fc and Ps (high Fs/low Fc/low Ps) or only one of them high (high Fs/high Fc/low Ps or high Fs/low Fc/high Ps); 2) "intermediate 1" characterized by low Fs with either high Fc or high Ps (low Fs/low Fc/high Ps or low Fs/high Fc/low Ps) (HR 3.368, 95% CI 1.488-7.626,P = 0.004); 3) "intermediate 2" characterized by high expression of all three markers (high Fs/high Fc/high expression levels of cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Sn), membrane E-cad (Em), membrane β-cat (Bm), and cytoplasmic Fascin (Fc) were combined with stromal PN (Ps) to investigate the prognostic strength (